Robotic pulmonary segmentectomy, initial experience in the Czech Republic.

INTRODUCTION: The initiation of lung cancer screening in Czechia and diagnosis in earlier stages has been reflected by an increasing demand for anatomical lung segmentectomy. The purpose of this study was to describe early results of the first robotic-assisted thoracoscopic segmentectomies performed in the country. METHODS: Our institution has performed 151 robotic anatomical lung resections since the initiation of the screening program in August 2020, which enabled us to attain the status of a proctoring and case observation centre. The robotic segmentectomy program was initiated after completing 70 robotic lobectomies. We performed a retrospective analysis of the results of our first 20 patients indicated for robotic segmentectomy. RESULTS: Median age of the patients was 60, with 11 females and 7 males. The most common indications included primary lung malignancy (n=13), pulmonary metastasis (n=2) and benign lesions (n=3). We performed 11 simple segmentectomies, 6 complex (S2, S3, S1a+2, S10 on the right) and one right S6 segmentectomy with bronchoplasty. The mean number of harvested lymph nodes for NSCLC was 20, the mean blood loss was 25 mL (from 10 mL to 100 mL), and the mean operative time was 200 minutes. All resection margins were tumour-free. There was no conversion to thoracotomy. Two patients were excluded as they required conversion to robotic lobectomy given that their lesions were localized close to the intersegmental plane. One complication (recurrent laryngeal nerve paralysis) occurred in 1 patient. Mean chest tube duration was 1.9 days and length of stay 3.9 days. CONCLUSION: Our experience suggests that implementation of the robotic segmentectomy program after completion of the robotic learning curve provides promising outcomes. Robotic technology and preoperative planning facilitate this technically demanding procedure especially when bronchoplasty is required.

Surgical therapy of chronic lung allograft dysfunction.

Lung transplantation has become a standardized and widely accepted treatment modality for selected end-stage lung diseases. Many factors influ- ence the long-term survival of patients after lung transplantation. One of the most important is clearly the development of chronic lung allograft dysfunction (CLAD). This review summarizes current knowledge of the histopathology of CLAD and its clinical characteristics. It also describes lung re-transplantation as the only causal therapy, its possible complications, and outcomes in standard and high-urgency patients awaiting a suitable organ with extracorporeal membrane oxygenation support. Fundoplication is an important surgical modality potentially leading to an improvement of the patients' condition. The indications and outcomes of this surgical procedure are discussed in a separate chapter. In addition, several nonsurgical treatment options aimed at slowing the progression of CLAD are outlined, as well as ongoing research focused on extending the life of these patients.

Biportal robotic pulmonary lobectomy, initial experience - case report.

INTRODUCTION: Thanks to perfect visualization and high maneuverability of instruments, the robotic technique is a preferable type of lung resection, even though the number of required incisions is usually higher compared to the video-assisted approach. This case report presents our initial experience with the reduced-port approach in performing robotic biportal lobectomy. CASE REPORT: The 72-years-old female, examined for hemoptysis, was diagnosed with a carcinoid tumor of the left lower lobe bronchus based on bronchoscopy. The patient underwent a biportal fully robotic left lower lobectomy. The time of operation was 235 minutes, longer compared to the average time of multiportal procedures, i.e. 190±52 minutes, and the blood loss of 100 mL was higher compared to 43±54 mL. The patient was discharged without complications on the third postoperative day. Histological analysis confirmed the diagnosis of a typical carcinoid with tumor free margins and seven tumor free lymph nodes. The patient continues to be followed at the Department of Pneumology, showing no signs of disease recurrence for eight months. CONCLUSION: The robotic biportal approach offers a reduction in chest wall traumatization while maintaining oncological radicality. Although this approach is safe and feasible, limitations in instrument movements necessitate specific training.

The first lung retransplantation with “ECMO bridge” in the Czech Republic - case report.

During the last 23 years of the National Lung Transplant Program in the Czech Republic, more than 500 lung transplantations, 4 retransplantations and one lobar retransplantation have been performed. We present the case report of a female patient with cystic fibrosis who underwent her first bilateral lung transplantation in January 2020. Due to a chronic lung allograft dysfunction, the patient required ECMO support and retransplantation. For the first time in the Czech Republic, a lung retransplantation with “ECMO bridge to (re)transplantation” preoperative support was performed in April 2021. The patient was discharged 39 days after retransplantation in a stable condition. At the day 90 follow-up visit, the patient was in a generally good condition with satisfying spirometric functions.

Video-assisted and robotic-assisted thoracoscopic pulmonary lobectomies, our experience.

INTRODUCTION: The use of video-assisted (VATS) and robotic-assisted (RATS) thoracoscopic surgery for anatomical pulmonary resections has been rapidly increasing. This study aimed to analyze our results of minimal invasive lobectomies to safely introduce these techniques to our practice. METHODS: Starting these new programs we followed the recommended steps including case observations and a proctoring. We retrospectively analyzed the data of our 7-year experience with VATS lobectomies and 1-year experience with RATS lobectomies. RESULTS: 128 minimal invasive lobectomies were performed between 4/2015 and 4/2021 in our center. The mean age of our patients was 64.7±10.5 years; 61 (47.7%) were women and 67 (52.3%) were men. Pulmonary malignancy was the main indication in 116 (90.6%) patients, including 2 patients with localized small cell lung cancer (SCLC). In 12 (9.4%) cases we operated for bronchiectasis and benign lung lesions. Stage I lung cancer was found in 57 (66.3%), stage II in 22 (25.6%) and stage III in 7 (8.1%) patients. We performed 110 VATS and 18 RATS lobectomies with a clear shift from triportal VATS to uniportal VATS and RATS in the last years. The mean operative time was 166±55.5 minutes and a conversion was approached in 8 (6.2%) cases (4 bleedings - less than 300 ml in all cases, 3 oncological cases, 1 case for a technical reason). The median postoperative length of stay was 4 days. CONCLUSION: VATS and RATS lobectomy has become a standard approach for early stages of lung cancer. Respecting the rules of introducing VATS and RATS including proctoring offers safety without any negative impact on survival or oncological radicality.

Starting the first robotic lobectomy program in the Eastern Europe during Coronavirus disease-2019 pandemic.

BACKGROUND: We report our experience in starting RATS (robotic-assisted thoracic surgery) lobectomy program during COVID-19 pandemic. METHODS: Data from 20 consecutive cases undergoing RATS lobectomy between August 2020 and April 2021 were prospectively accumulated into our database. RESULTS: The mean operational time was 235±69 minutes (median 210, range 175 to 370). Conversion-to-open rate was 5 %. One patient was converted to an open procedure during surgery due to surgical bleeding. One patient (5 %), with sever chronic obstructive pulmonary disease (COPD), had prolonged air leak with chest drainage 11 days and conservative treatment. Morbidity rate was 10 % (2 patients). Estimated costs of RATS lobectomy in our department were $9,590 (range $8,250-$12,730). 30-days mortality was 0%. CONCLUSIONS: Safe robotic surgery is based not only on improved robotic equipment, but also on good technical skills and medical knowledge. It requires training of the entire operating room team. The learning curve is steep, involving port placement, use of the correct robotic arms, availability of the proper instrumentation, and proper patient positioning (Tab. 2, Ref. 28).

SARS-CoV-2 viral load assessment in lung transplantation.

In the era of COVID-19 pandemic, organ transplantation programs were facing serious challenges. The lung transplantation donor pool was extremely limited and SARS-CoV-2 viral load assessment has become a crucial part of selecting an optimal organ donor. Since COVID-19 is a respiratory disease, the viral load is thought to be more important in lung transplantations as compared to other solid organ transplantations. We present two challenging cases of potential lung donors with a questionable COVID-19 status. Based on these cases, we suggest that the cycle threshold (Ct) value should always be requested from the laboratory and the decision whether to proceed with transplantation should be made upon complex evaluation of diverse criteria, including the nasopharyngeal swab and bronchoalveolar lavage PCR results, the Ct value, imaging findings and the medical history. However, as the presence of viral RNA does not ensure infectivity, it is still to be clarified which Ct values are associated with the viral viability. Anti-SARS-CoV-2 IgA antibodies may support the diagnosis and moreover, novel methods, such as quantifying SARS-CoV-2 nucleocapsid antigen in serum may provide important answers in organ transplantations and donor selections.

Malignancy after lung transplantation.

INTRODUCTION: Lung transplantation has become a successful life-saving treatment for patients with end-stage pulmonary disorders. Long-term survival outcomes after lung transplantation have been improving with increasing experience. Malignancies occupy the third position among the causes of death, particularly between years 5 to 10 from lung transplantation. The risk factors include predominantly high doses of immunosuppressive therapy, older age, infections caused by oncogenic viruses and smoking history. METHODS: We retrospectively evaluated all patients undergoing lung transplantation between 2010 and 2019. The aim of this study was to analyze the incidence, type and location of tumors, time from detection, survival time and cause of death in patients with malignant tumors after lung transplantation. RESULTS: In total, 308 lung transplantations were performed at the 3rd Department of Surgery of the 1st Faculty of Medicine, Charles University and University Faculty Hospital in Motol between 2010 and 2019. Posttransplant malignancy was diagnosed in 32 patients; a tumor was detected in the explanted lung in 5 patients. Lung cancer was the most frequent tumor in our study and was found in 13 patients (37%); 6 patients (17%) had a nonmelanoma skin cancer; and posttransplant proliferative disease developed in 4 patients (12.5%). The incidence rate of other types of malignancy was low. Mean of survival after diagnosis was 152 days. CONCLUSION: Life time administration of immunosuppressive therapy in lung transplanted patients plays a key role in the prevention of rejection but on the other hand it represents a risk factor for cancer development. Oncological management of posttransplant cancer is based on reduction of immunosuppressive therapy, combined with surgical resection of solid organ tumors and other types of cancer therapy. Oncology screening tests should be done regularly as a method of prevention, and for an early detection of any tumor.

Survivin, a novel target of the Hedgehog/GLI signaling pathway in human tumor cells.

Survivin, an important antiapoptotic protein, is expressed in tumors, whereas in normal tissues the expression of this protein is extremely low, defining a role for survivin as a cancer gene. Survivin exhibits multifunctional activity in tumor cells. However, why survivin expression is sharply and invariably restricted to tumor tissue remains unclear. Here, we identified 11 putative consensus binding sites for GLI transcription factors in the survivin promoter and characterized the promoter activity. Inhibitors of the Hedgehog/GLI pathway, cyclopamine and GANT61, decreased the promoter activity in reporter assays. ΔNGLI2 (which lacks the repressor domain) was the most potent vector in activating the survivin promoter-reporter. Moreover, GANT61, a GLI1/2 inhibitor, repressed endogenous survivin protein and mRNA expression in most cells across a large panel of tumor cell lines. Chromatin immunoprecipitation showed GLI2 binding to the survivin promoter. The ectopic GLI2-evoked expression of endogenous survivin was observed in normal human fibroblasts. GANT61 decreased survivin level in nude mice tumors, mimicking the activity of GANT61 in cultured cells. The immunohistochemistry and double immunofluorescence of human tumors revealed a correlation between the tissue regions showing high GLI2 and survivin positivity. Thus, these results demonstrated that survivin is a classical transcriptional target of GLI2, a Hedgehog pathway signaling effector. This potentially reflects the high expression of survivin in human tumor cells. As the Hedgehog pathway is upregulated in virtually all types of cancer cells, these findings substantially contribute to the explanation of uniform survivin expression in tumors as a potential target for the development of a more effective treatment of cancers through the inhibition of GLI2 to restrain survivin activity.

Distinct co-regulation of endogenous versus transfected MITF-dependent tyrosinase promoter.

The tissue-specific control of gene activation in melanocytes is directed by the microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte development and differentiation. Tyrosinase is a rate-limiting enzyme in melanin biosynthesis and a prototypic MITF target. While the expression of tyrosinase is restricted to pigmented cells, the transfected tyrosinase promoter is active in a broad range of cell types if ectopic MITF is co-expressed. Here we used the E1A oncoprotein and its mutants as repressors of both the transiently transfected and endogenous tyrosinase promoter. We report that the requirement of the E1A N-terminus for repression of the MITF-activated tyrosinase promoter and the sensitivity to derepression by the histone deacetylase inhibitor trichostatin A are distinct when the activity of the transiently transfected or the endogenous promoter is analysed in U2-OS cells. Thus, for transiently transfected versus chromatin-embedded promoter, the activity of obligatory MITF seems to be executed through different mechanisms of transcriptional coactivation.

Low mutational rate of K-ras codon 12 in singular bronchoscopy specimens in suspected lung cancer.

Mutations of the K-ras gene are found in a subset of non-small- cell lung carcinomas (NSCLC). The aim of our study was to determine the K-ras codon 12 mutation in the first, singular bronchoscopy specimen in parallel with the cytological examination for the diagnosis of lung cancer. Samples were obtained by diagnostic bronchoscopy in 140 patients with suspected lung tumors. The analysis of K-ras mutations was carried out by a sensitive two-step mutation- enriched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. This method has been confirmed earlier to be positive for mutated tumor cells and negative for normal lung parenchyma and bronchus. Of the 140 patients with suspected cancer, 93 were diagnosed as NSCLC by cytology or histology in either the same specimen used for the detection of K-ras mutation or in later biopsies. However, only four K-ras codon 12 mutations were detected in the first bronchoscopic material: one in adenocarcinoma, two in squamous cell tumors, and one mutation was found in a patient with dysplasia which was diagnosed later as a squamous cell carcinoma. Our findings indicate that although the K-ras (codon 12) mutation is a gene lesion infrequently detectable in a singular specimen taken at the first bronchoscopy examination in cases of clinically suspected lung cancer, the detection of this mutation can help to confirm the cytological diagnosis of NSCLC or may be even diagnostic in cytologically negative cases.

Repression of the melanocyte-specific promoter of the microphthalmia-associated transcription factor by the adenoviral E1A 12S oncoprotein.

Melanocytes express MITF, which is crucial for the development of the melanocyte lineage and is overexpressed in malignant melanomas. Adenoviral E1A protein-expressing melanocytes are unpigmented, with the expression of MITF being silenced. We tested here a direct repression of the melanocyte-specific MITF promoter by E1A and its mutants. We found that the extreme N-terminus and conserved region 1 are required for repression. In contrast, the motif in conserved region 2 (a.a. 122-126), as well as amino acids 26-35 at the N-terminus, are not necessary. As these two later motifs mediate E1A binding to the retinoblastoma protein or to the transcriptional co-activator TRRAP, respectively, and are important for transformation by E1A in cooperation with other oncogenes, the results suggest that the transformation-defective E1A can still efficiently repress the MITF promoter. The CREB binding motif-mutated promoter had lower activity, but was also repressed by the same E1A mutants in human melanoma cells. The E1A protein is known to also exert an antitumour activity, which is associated with its transcription repression function and the ability to induce apoptosis, and is a potential antimelanoma agent. Since recent data suggest that MITF may be a survival factor for melanoma cells, the E1A mutants described here might constitute a good targeting agent for antimelanoma therapy.

Microphthalmia transcription factor: a specific marker for malignant melanoma.

The transcription factor microphthalmia (MITF) is required for the formation of normal melanocytes during embryonic development and for the expression of pigment cell-specific markers, which are the downstream transcriptional targets of MITF. It also seems to be crucial for the survival of malignant melanocytes. The special interest of this review is the possible utility of MITF as a marker of malignant melanoma. Melanocyte-specific isoform of MITF appears to be a unique molecule in the differential diagnosis of melanocytic tumors.

[Complete atrioventricular block (with Adams-Stokes syndrome) in systemic connective tissue disease. Electrocardiographic development in three patients]. / Uplná sínokomorová blokáda (s Adams-Stokesovým syndromem) u systémových onemocnení pojivové tkáne. Elektrokardiografický vývoj u trí pozorovaných nemocných.

The association of high grade atrioventricular heart block with systemic connective tissue diseases is very rare. To date, only sporadic case reports or reviews appeared in the literature. Three cases of such association observed by the author are described here. The patients were a 51 years old man with systemic sclerosis and two women, a 64 years old patient with visceral nodous seropositive rheumatoid arthritis, and second was a 74 years old patient with systemic lupus erythematosus, the oldest of the group of patients with this disease. In two of the three patients, Adams-Stokes attack was a cause of death. The author did not observe this high grade atrioventricular block in patients with systemic connective tissue diseases the last 32 years. First and second grade heart blocks were, however, o chi asionally seen and responded well to the treatment of the systemic disease. Thus, in patients with systemic connective tissue diseases and emergency symptoms, it is recommended to consider also this rare association. While, two decades ago, patients frequently died after the Adams-Stokes attack, the treatment of the high grade block is now successful due the permanent cardiostimulator. In the last years, there are almost no reports about the association of systemic connective tissue diseases with high grade heart block, presumably because of the efficient new treatment approached to systemic diseases including modern immunomodulation drugs.

Transcriptional repression of the microphthalmia gene in melanoma cells correlates with the unresponsiveness of target genes to ectopic microphthalmia-associated transcription factor.

In the melanocyte, expression of genes required for pigment formation is mediated by the microphthalmia transcription factor, which is also critical for the development and survival of normal melanocytes during embryogenesis. Here we show that the expression of the melanocyte-specific isoform of microphthalmia transcription factor is lost in a subset of human melanoma cell lines, accompanied by the repression of tyrosinase and tyrosinase-related proteins 1 and 2, the three transcriptional target genes for microphthalmia. After the forced expression of microphthalmia transcription factor in melanoma cells where the expression of endogenous microphthalmia gene was found to be extinguished, no restoration of the melanogenic phenotype occurred and the transcription of the three microphthalmia transcription factor target genes remained silent. The transcription activation domain of microphthalmia transcription factor, tested as a GAL-MITF fusion protein, remained fully functional in these cells, however, and ectopic microphthalmia transcription factor localized normally to the nucleus and bound to the tyrosinase initiator E-box in gel retardation assays. Thus, the block of differentiation in microphthalmia-transcription-factor-negative melanomas extended the transcriptional repression of the microphthalmia transcription factor gene alone, and endogenous promoters in these melanoma cells became no longer responsive to microphthalmia transcription factor when this was substituted exogenously. The data presented suggest that a specific nuclear context is required for the transcriptional activation of the melanocyte markers by the microphthalmia transcription factor in malignant melanocytes and this specificity is lost concomitantly with the transcriptional repression of microphthalmia transcription factor.

Expression of genes for microphthalmia isoforms, Pax3 and MSG1, in human melanomas.

Microphthalmia (MITF) gene product, a transcription factor of the basic-helix-loop-helix type, is thought to play a role in the regulation of genes encoding the enzymes necessary for melanogenesis. These include tyrosinase, TRP-1 and TRP-2. Melanocyte-specific isoform of microphthalmia, MITF-M, is expressed in normal and malignant melanocytes. The presence of two other isoforms of microphthalmia, MITF-A and MITF-H, which differ from MITF-M in the amino-terminus, was demonstrated also in some non-melanocytic lineages. Here we have analyzed the presence of all three known isoforms of MITF mRNA in a panel of 17 human melanoma cell lines by a reverse transcriptase-polymerase chain reaction using isoform-specific primers. While, as expected, the predominant form in melanoma cell lines was MITF-M, low amounts of MITF-A mRNA was found in almost all melanomas, as well as in most of 20 tumor cell lines of the non-melanocyte origin (lung and colon carcinomas, osteosarcomas and neuroblastomas). The expression of MITF-H was not detected, with a few exceptions, in the tested cell lines. Pax3 transcription factor was reported earlier to regulate positively the melanocyte-specific promoter of the MITF gene. We found here that the Pax 3 mRNA was expressed in all melanoma cell lines, even in those that had repressed the MITF-M and were amelanotic. This suggests that additional factors, besides Pax3, are required for the MITF expression. The MSG1 (melanocyte-specific gene 1), a gene originally isolated from melanocytes and containing a strong transcription activation domain, was also found expressed in all melanomas and most non-melanocyte tumor cell lines. Together, these data indicate that the MITF-M isoform is the major type of MITF mRNA present in human melanoma cell lines and show that the expression of the isoform MITF-A and the MSG1 is not restricted to malignant melanocytes and occurs in a wide range of tumor cell lines.

Occurrence of ras mutations in human lung cancer. Minireview.

Members of ras family of oncogenes, when activated by a point mutation, have been implicated in many types of human cancers. In several types of human solid tumors, point mutations of the K-ras gene are relatively frequent. Among lung cancers, a subset of non-small cell lung carcinomas, mostly adenocarcinomas, contains activated K-ras. The examination of K-ras mutations in samples obtained for diagnostic reasons, such as bronchial biopsies or bronchoalveolar lavage fluid, may be used as a supplement in the early diagnosis of lung adenocarcinoma.

Expression of the aromatic L-amino acid decarboxylase mRNA in human tumour cell lines of neuroendocrine and neuroectodermal origin.

Neuroendocrine differentiation of lung tumours is characterised by the expression of several neuroendocrine markers and is confined mostly to specific histological subtypes, i.e. small cell carcinomas and carcinoids. One of the markers seen in neuroendocrine tumours, high activity of the aromatic L-amino acid decarboxylase (AADC), is helpful in distinguishing the classic and variant small cell lung tumour subtypes. Here, we have analysed the expression and quantified the level of mRNA coding for AADC in human tumour cell lines by use of the reverse transcription and polymerase chain reaction (RT-PCR). High amounts of mRNA were detected in classic small cell lung carcinomas and a neuroblastoma cell line. Other cell lines (melanomas, non-small cell lung carcinomas and osteosarcoma) also showed AADC expression, but the levels were 2-3 orders lower. Also, the tissue-specific (neuronal versus liver-specific) mRNA type has been estimated. Small cell lung carcinomas, neuroblastoma and melanoma expressed messenger RNA specific for neuronal tissues. Importantly, the non-small cell lung carcinoma cell lines expressed either liver-specific (non-neuronal) mRNA (cell line A549) or predominantly the neuronal (cell line NCI-H520) AADC message. These data indicate that a range of tumour cell lines transcribe the AADC gene and that two distinct types of AADC mRNA which reflect the embryonal (neuronal or non-neuronal) origin of the tumour may be produced in non-small cell lung cancer cells.